WO2024047009A1

WO2024047009A1 - Methods and uses for modifying gut flora in aquatic species

Info

Publication number: WO2024047009A1
Application number: PCT/EP2023/073601
Authority: WO
Inventors: Rutchanee CHOTIKACHINDA; Ester SANTIGOSA CULI
Original assignee: Novozymes AS; DSM IP Assets BV
Current assignee: Novozymes AS; DSM IP Assets BV
Priority date: 2022-08-30
Filing date: 2023-08-29
Publication date: 2024-03-07
Anticipated expiration: 2025-02-28
Also published as: EP4580656A1; CL2025000521A1

Abstract

The present application concerns methods and uses for improving the balance of beneficial and deleterious bacteria in the gastrointestinal tract of aquatic species. It also concerns a method of culturing an aquaculture of an aquatic species, comprising improving the balance of beneficial and deleterious bacteria in the gastrointestinal tract of said aquatic species by the means of a composition comprising a protease.

Description

Methods and uses for modifying gut flora in aquatic species

Around the world, the demand for seafood has increased greatly because people have learned that the seafood in regular diet confer health benefits. Aquaculture provides almost half of the world's fish for human consumption. The growth rate of worldwide aquaculture has been sustained and rapid, averaging about 8 percent per annum for over thirty years, while the take from aqua cultures has been essentially flat for the last decade.

However, intensive culture of fish and crustaceans, especially, intensive shrimp culture has led to outbreaks of various diseases, resulting in annual economic losses to the aquaculture industry estimated at billions of dollars worldwide. Major pathogens that are affecting the aquaculture industry include: bacteria, fungi, viruses and parasites. Given that bacteria can survive well in aquatic environment independently of their hosts, bacterial diseases have become major impediments to the aquaculture. For instance, disease is now considered to be the major limiting factor in the shrimp culture sector.

Vibrio species includes some of the most potent pathogens. They can cause bacterial infections in fish and shrimp aquaculture farms, and can cause infection and toxicity as a primary problem and production of biofilms and bioluminescence as a secondary problem.

In the past, varieties of measures were taken to subside and/or eliminate deleterious effects caused by such bacteria. For example, use of antibiotics and/or disinfectants, improvement of hygiene and bio-security, production of immune stimulants such as glucans and the like. Although several of these means could partly solve the problem, most of them are unsustainable for a long term application. In particular, there is a growing concern about the use of antibiotics in aquaculture due to development of resistance.

Feeding and new practices in farming usually play an important role in aquaculture, and the addition of various additives, such as enzymes to a balanced feed formula to achieve better growth is a common practice of many fish and shrimp feed manufacturers and farmers. More recently, utilization of probiotic(s) for biological control of one or more pathogens targeting their virulence (e.g. quorum sensing) has caught significant interest of the researchers. Probiotics, as ‘biofriendly agents’ such as lactic acid bacteria and Bacillus spp., can be introduced into the culture environment to control and compete with pathogenic bacteria as well as to promote the growth of the cultured species. Nevertheless, culture of aquatic species constantly needs new techniques in order to increase production yield and maintain healthiness in aquatic species. Indeed, although adding probiotic to feed for aquatic species is helpful, it would be desirable to influence the aquatic species’ microbiome such that it harbours more beneficial rather than potentially deleterious bacteria.

There thus remains a need in the art for new methods of modifying gut flora, especially adjusting the balance of beneficial and deleterious (e.g., pathogenic) bacteria in the gut in favour of beneficial species, in aquatic species. The underlying technical problem of this application is to fulfil this need.

The technical problem is solved by the subject-matter as defined in the claims, described in the description, exemplified in the Examples and illustrated in the Figures.

The inventors surprisingly found that feeding aquatic species with a composition comprising a protease, such as a serine protease improves the balance of beneficial and deleterious bacteria in the gastrointestinal tract of aquatic species. This is indeed surprising, since thus far, enzymes, such as a proteases were added to, e.g. feed for aquatic species with the aim of improving digestion of feed and, thus, improving feed efficiency. However, the effect observed by the present inventors was neither thought of nor seen before in the prior art.

As is shown in Example 2 and illustrated in Figure 6, shrimps fed with a protease, e.g., 300 g protease per 1 ton of feed, have a reduced amount of Vibrio species, being an example of deleterious bacteria for aquatic species, while the amount of Lactobacillus species increased (see Figure 6, and compare 1st bar with 4th bar). This effect was not seen in the control which was not fed with the same protease (see 4th bar). On the contrary, in the negative control (not fed with a protease), the least amount of Lactobacillus species and the most amount of Vibrio species is seen (see 4th bar). Yet, the massive amount of Vibrio species is reduced, when a protease is added (see 4th to 6th bar). It can also be seen that the amount of protease, i.e. , more protease fed to aquatic species enhances the amount of Lactobacillus species (see 1st to 3rd bar), while decreasing the amount of Vibrio species (see 4th to 6th bar).

As is known, vibriosis is caused by an infection of Vibrio bacteria species. Vibrio species are present at all times within the aquatic environment or may enter an aquatic culture system if strict quarantine practices for new species or sick species may be ignored. Immunocompromised aquatic species, such as those stressed by poor water quality, bullying, or lack of sufficient nutrition, are more susceptible to bacterial infection. Traumatic wounds on an aquatic species are also potential areas where there can be increased bacteria growth. Vibrio bacteria are known to be more problematic in warmer temperatures. Vibriosis can result, e.g. in a fish developing skin ulcerations and hemorrhaging because of the liquefying of its internal organs which can show up as redness and ulcerations of the skin, face, fin, and tail. The results are redness throughout the body, fins, tail, eyes, and mouth.

In order to cope with deleterious effects, e.g. caused by Vibrio species different measures are applied in the art. Among these measures is the use of probiotics, such as Lactobacillus species, as feed additive for feeding aquatic species. Hence, it is plausible that beneficial bacteria, such as Lactobacillus species, contribute to deal with deleterious bacteria, such as Vibrio species.

In line with this, Anas et al. (Biotech. 2021 Feb;11(2):66; doi: 10.1007/s13205-020-02618-2) reports that lactic acid bacteria have probiotic potential to prevent vibriosis disease outbreaks in shrimp aquaculture systems. These lactic acid bacteria were isolated from gut of shrimps and shown to have antagonistic activity against Vibrio parahaemolyticus and Vibrio campbellt.

Vieira et al. (Brazilian Journal of Oceanography 2007, 55(4); doi:10.1590/S1679- 87592007000400002) reports that lactic acid bacteria increase the survival of marine shrimp, after infection with Vibrio harveyi.

Zheng et al. (Aquae Res 2017, 48: 2767-2777; doi.org/10.1111/are.13110) reports that lactic acid bacteria have a posiive effect on growth performance of shrimp.

Gao et al. (Fish Shellfish Immunol. 2016 Jul;54:573-9; doi: 10.1016/j.fsi.2016.05.013) reports Lactobacillus plantarum lipoteichoic acid has potential as a therapeutic agent against V. anguillarum-caused vibriosis in fish.

Moeller et al. (J Mar Syst 2021 , Vol. 221 :103574; doi. org/10.1016/j.jmarsys.2021.103574) reports that fish serve as a winter reservoir for Vibrio ssp. in the southern Baltic Sea coast.

Ringo et al. (Front. Microbiol. 9: 1818; doi: 10.3389/fmicb.2018.01818) reports on lactic acid bacteria in finfish and states that the Gl tract in fish is one of the most important interfaces with the environment exposed to potential pathogens, it is of importance to evaluate the presence of beneficial bacteria such as lactic acid bacteria in the Gl tract, as autochthonous bacteria rapidly colonize the digestive tract at early developmental larval stages of finfish. The authors thus conclude that that lactic acid bacteria administration results in beneficial effects such as disease resistance and weight gain in finfish aquaculture.

Bearing in mind that the prior art constantly reports that beneficial bacteria, such as Lactobacillus species have a positive effect on aquatic species, e.g. for their health or growth, etc., it is reasonable that, when the present invention improves the balance of beneficial and deleterious bacteria in the gastrointestinal tract of aquatic species, such improvement has a positive effect on, e.g. health and/or growth of aquatic species, e.g. those referred to herein as being preferred. Put differently, the clue of the present invention is to improve the balance between beneficial and deleterious bacteria in the gastrointestinal tract of aquatic species by feeding aquatic species with a composition comprising a protease, rather than feeding aquatic species with beneficial bacteria, e.g. Lactobacillus species. Of course, the present invention does not exclude feeding aquatic species in addition to a composition comprising a protease as described herein with one or more probiotic bacteria.

However, the surprise that came with the present invention is the finding that the level beneficial bacteria can be increased by feeding aquatic species with a composition comprising a protease, rather than merely feeding aquatic species with beneficial bacteria. This effect could not have been suspected, let alone expected.

Without being bound by theory, the present invention provides thus for more sustainability, since the level of beneficial bacteria can be increased in the gut - most likely they settle and can remain in the gut, which is more sustainable than just being added beneficial bacteria through feed, whereby such externally added beneficial bacteria may be not able to settle and remain in the gut. Thus, the sustainability is assumed to reside in the fact that aquatic species have in the long run a benefit from the increase of the level of beneficial bacteria caused - as is shown in the Examples - by an exogenously fed protease, rather than by constantly receiving beneficial bacteria through feed which may no be able to settle and grow in the gut of the aquatic species. Indeed, thus far, no such effect as was observed and proven by the present inventors was known, because protease would never have been thought to be the causative agent improving the balance of beneficial and deleterious bacteria in the gut of aquatic species. That is to say - the prior art had merely the obvious thing in mind, i.e., feed aquatic species with beneficial bacteria, but not feed aquatic species with a protease which causes an increase in the level of beneficial bacteria, while decreasing deleterious bacteria. However, the present inventors took the non-obvious action and fed aquatic species with a protease, thereby observing and proofing that the level of beneficial bacteria is enhanced, while the level of deleterious bacteria is decreased, i.e., the balance between the level of beneficial and deleterious bacteria in the gut of aquatic species is improved.

Accordingly, the present invention relates to the use of a composition comprising a protease for improving the balance of beneficial and deleterious bacteria in the gastrointestinal tract of aquatic species. Also, the present invention relates to a method for improving the balance of beneficial and deleterious bacteria in the gastrointestinal tract of aqua species, comprising feeding aquatic species with a composition comprising a protease.

Furthermore, the present invention relates to a method of culturing an aquaculture of an aquatic species, comprising improving the balance of beneficial and deleterious bacteria in the gastrointestinal tract of said aquatic species by the means of a composition comprising a protease.

The “balance of beneficial and deleterious bacteria” as used herein is equivalent to the longer phrase “balance of the level of beneficial and deleterious bacteria”. As described herein below, the skilled person can determine such “level” which, as used herein, is equivalent to an “amount”. Thus, the longer phrase as described before may also read “balance of the amount of beneficial and deleterious bacteria”.

The aquatic species referred to in the methods and uses of the present invention are preferably selected from crustaceans or fish. Preferably, the fish referred to in the methods and uses of the present invention are warm water fish or cold water fish. The warm fish are preferably selected from tilapia, seabream, seabass, or carp. The cold water fish are preferably selected from salmon or rainbow trout.

Preferably, the crustaceans referred to in the methods and uses of the present invention are shrimps.

Of note, it is preferred that the methods and uses of the present invention are non- therapeutic. Indeed, the improvement of the balance of beneficial and deleterious bacteria in the gastrointestinal tract of aquatic species is deemed to contribute to, e.g. the health or growth of the aquatic species which is by way of common sense non-therapeutic.

Accordingly, it is preferred in the context of the methods and uses of the present invention that the aquatic species are healthy.

When used herein, the term “gut” is equal to the term “gastrointestinal tract”. Thus, one term can replace the other and, vice versa.

The gut flora, i.e., the community of bacteria resident in the gastrointestinal tract, comprises both beneficial and deleterious bacterial types or species. Whether a particular member of the gut flora is beneficial, deleterious or inconsequential to the health of the aquatic species in particular circumstances can depend on a number of factors, but for the purposes of the present invention certain types or species of bacteria can be considered beneficial and others deleterious. Examples of beneficial members of the gut flora include bifidobacteria (species of the genus Bifidobacterium) and lactic acid bacteria, more particularly species of the genus Lactobacillus.

Deleterious bacteria include pathogenic bacteria. Examples of deleterious members of the gut flora include Vibrio ssp., Clostridium spp., Desulfovibrio spp., Helicobacter spp. or pathogenic forms of Escherichia coli.

Gastrointestinal health typically depends on maintenance of an appropriate balance of beneficial and deleterious bacteria. An increase in the level of deleterious bacteria and/or a decrease in the level of beneficial bacteria can be associated with a decline in gastrointestinal health. Conversely, an increase in the level of beneficial bacteria and/or a decrease in the level of deleterious bacteria can be associated with an improvement in gastrointestinal health, for example improvement of health.

“Improvement” or “improving” or other grammatical forms include "enhancing" or "enhancement" of the balance between the level of beneficial and deleterious bacteria as described herein. An “improvement” herein means shifting the balance in favor of the level of beneficial bacteria, and thus can involve an increase in the level beneficial bacteria and/or a decrease in the level deleterious bacteria. In some embodiments of the invention, enhancement of the balance arises from both a reduction in the level of deleterious, e.g. pathogenic, bacteria and an increase in the level of beneficial bacteria.

An increase in the level beneficial bacteria in the gut of an aquatic species fed with a composition comprising a protease as described herein encompasses an increase of 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 250% or more in the level of beneficial bacteria in the gut of the aquatic species in comparison to an aquatic species not fed with a composition comprising a protease as described herein. Indeed, as is shown in Example 2, the increase in the level of beneficial bacteria between the positive control and the negative control is about 100 to 200% in, e.g. Lactobacillus species. Accordingly, an increase in the level of beneficial bacteria in the context of the methods and uses of the present invention may advantageously be 100%, 125%, 150%, 175%, 200% or more.

An decrease in the level of deleterious bacteria in the gut of an aquatic species fed with a composition comprising a protease as described herein encompasses a decrease of 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 250% or more in the level of deleterious bacteria in the gut of the aquatic species in comparison to an aquatic species not fed with a composition comprising a protease as described herein. Indeed, as is shown in Example 2, the decrease in the level of deleterious bacteria between the positive control and the negative control is about 100 to 200%. Accordingly, an decrease in the level of deleterious bacteria in the context of the methods and uses of the present invention may advantageously be 100%, 125%, 150%, 175%, 200% or more.

Bacterial populations in the gut flora can be estimated by any procedure known in the art. For example, feces samples can be cultured using traditional plating methodologies, or illustratively by the fluorescence in situ hybridization (FISH) technique.

The term "aquaculture" as used in the present disclosure generally relates to aqua-farming, the farming of aquatic species such as fish or crustaceans in variety of environments including but not limited to tanks, lakes, ponds, or any other natural or man-made aquatic reservoirs that can be suitable for breeding, hatchery, rearing and harvesting of the aquatic species.

Preferably, in the methods and uses of the present invention, the improvement of the balance of beneficial and deleterious bacteria in the gastrointestinal tract of aquatic species comprises an increase in the level of beneficial bacteria and a decrease in the level of deleterious bacteria.

Preferably, the beneficial bacteria comprise one or more of lactic acid bacteria, preferably Lactobacillus spp. or the deleterious bacteria comprise one or more of Vibrio ssp.

Preferably, the beneficial bacteria comprise one or more of lactic acid bacteria, preferably Lactobacillus spp. and the deleterious bacteria comprise one or more of Vibrio ssp.

When referred in the context of the methods and uses of the present invention, the composition is preferably a nutritional supplement, prebiotic or aquaculture inoculant which comprises a protease as described herein. The composition, however, may also be a complete feed for aquatic species which comprises a protease as described herein.

Preferably, the composition applied in the methods and uses of the present invention further comprises a carrier.

Preferably, the composition applied in the methods and uses of the present invention may further comprise one or more probiotic bacteria, preferably lactic acid bacteria, e.g. Lactobacillus spp. bacteria. Probiotics of interest herein comprise at least one kind of beneficial bacteria, for example bifidobacteria and/or lactic acid bacteria. In one embodiment a probiotic useful herein comprises beneficial bacteria comprising one or more of Bifidobacterium spp. and Lactobacillus spp. Preferably, the protease comprised by the composition referred to in the methods and uses of the present invention is preferably a serine protease, such as a Ronozyme ProAct. Ronozyme ProAct is a serine protease. It is preferably obtained or obtainable from Nocardiopsis sp.. In particular, it can be characterized in that it is derived from Nocardiopsis sp. NRRL 18262, and/or from Nocardiopsis alba (taxonomy based on Berge's Manual of Systematic Bacteriology, 2nd edition, 2000, Springer (preprint: Road Map to Bergey's)). It can also be characterized in that it is an acid-stable serine protease obtained or obtainable from Nocardiopsis dassonvillei subsp. dassonvillei DSM 43235 (A1918L1), Nocardiopsis prasina DSM 15649 (NN018335L1), Nocardiopsis prasina (previously alba) DSM 14010 (NN18140L1), Nocardiopsis sp. DSM 16424 (NN018704L2), Nocardiopsis alkaliphila DSM 44657 (NN019340L2) and Nocardiopsis lucentensis DSM 44048 (NN019002L2), as well as homologous proteases therefrom. In general, the term serine protease refers to serine peptidases and their clans as defined in the Handbook of Proteolytic Enzymes, A. J. Barrett, N. D. Rawlings, J. F. Woessner (eds), Academic Press (1998). In the 1998 version of this handbook, serine peptidases and their clans are dealt with in chapters 1-175. Serine proteases may be defined as peptidases in which the catalytic mechanism depends upon the hydroxyl group of a serine residue acting as the nucleophile that attacks the peptide bond. Examples of serine proteases for use according to the invention are proteases of Clan SA, e. g. Family S2 (Streptogrisin), e. g. Sub-family S2A (alpha-lytic protease), as defined in the above Handbook.

In addition or alternatively, the protease applied in the methods and uses of the present invention can be characterized in that it is (a) a polypeptide having a sequence identity of at least 70%, 75%, 80%, 85%, 90% or 95% to any one of SEQ ID NOs 1-5, with SEQ ID NO: 1 or 4 being preferred; (b) a variant of any one of SEQ ID NOs: 1-5, with SEQ ID NO: 1 or 4 being preferred, wherein the variant has protease activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 positions; (c) a polypeptide comprising the polypeptide of (a) or (b) and a N-terminal and/or C-terminal His-tag and/or HQ-tag; (d) a polypeptide comprising the polypeptide of (a) or (b) and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; or (e) a fragment of the polypeptide of (a) or (b) having protease activity and having at least 90% of the length of the mature polypeptide. Preferably, the protease comprised by the composition referred to in the methods and uses of the present invention has the amino acid sequence shown in SEQ ID NO: 1 , 2, 3, 4 or 5, with SEQ ID NO: 1 or 4 being preferred.

When reference is made herein to a SEQ ID NO., it is meant that reference is made to the amino acid sequence shown in the respective SEQ ID NO. Accordingly, a polypeptide when referred to herein has an amino acid sequence, e.g. shown in any one of the SEQ ID NOs. as referred to herein. Proteases applied in the methods and uses of the present invention have preferably the following amino acid sequences, with SEQ ID NO: 1 or 4 being preferred.

SEQ ID NO: 1

ADIIGGLAYTMGGRCSVGFAATNAAGQPGFVTAGHCGRVGTQVTIGNGRGVFEQSVFPGN DAAFVRGTSNFTLTNLVSRYNTGGYAAVAGHNQAPIGSSVCRSGSTTGWHCGTIQARGQS VSYPEGTVTNMTRTTVCAEPGDSGGSYISGTQAQGVTSGGSGNCRTGGTTFYQEVTPMV NSWGVRLRT

SEQ ID NO: 2

ADIIGGLAYTMGGRCSV

SEQ ID NO: 3

EVTATPSTQTPWGI KSI YN DQSITKTTGGSGI KVAVLDTGVHTGH I DLAGSSEQCKDFTQSN PLVNGSCTDRQGHGTHVAGTVLAHGGSDGQGVYGVAPQAKLWAYKVLGDNGSGYSDDIA AAIRHVADEASRTGSKVVINMSLGSSGKDSLIASAVDYAYGKGVLIVAAAGNSGSGSNTIGY PAALVNAVAVAALENVQQNGTYRVANFSSRGNPATAGDFRIQERDVEVSAPGASVESTWY NGGYNTISGTSMATPHVAGLAAKIWSSNSSLSHSQLRTELQNRAKVYDIKGGIGAGTGDDY ASGFGYPRVK

SEQ ID NO: 4

AVPSTQTPWGI KSI YN DQSITKTTGGSGI KVAVLDTGVYTSHLDLAGSAEQCKDFTQSNPLV DGSCTDRQGHGTHVAGTVLAHGGSNGQGVYGVAPQAKLWAYKVLGDNGSGYSDDIAAAI RHVADEASRTGSKVVINMSLGSSAKDSLIASAVDYAYGKGVLIVAAAGNSGSGSNTIGFPGG LVNAVAVAALENVQQNGTYRVADFSSRGNPATAGDYIIQERDIEVSAPGASVESTWYTGGY NTISGTSMATPHVAGLAAKIWSANTSLSHSQLRTELQNRAKVYDIKGGIGAGTGDDYASGFG YPRVK

SEQ ID NO: 5

AVPSTQTPWGIKSIYNDQSITKTTGGKGIKVAVLDTGVYTSHLDLAGSAEQCKDFTQSNPLV DGSCTDRQGHGTHVAGTVLAHGGSNGQGVYGVAPQAKLWAYKVLGDKGEGYSDDIAAAI RHVADEASRTGSKVVINMSLGSSAKDSLIASAVDYAYGKGVLIVAAAGNEGPKPNTIGYPAG FVNAVAVAALENVQEKGTYRVADFSSRGNPATAGDYIIQERDIEVSAPGASVESTWYTGGY NTISGTSMATPHVAGLAAKIWSANTSLSHSQLRTELQNRAKVYDIKGGIGAGPGDDYASGF

GYPRVK

Protease activity can be measured using any assay, in which a substrate is employed, that includes peptide bonds relevant for the specificity of the protease in question. Examples of protease substrates are casein, and pNA-substrates, such as Suc-AAPF-pNA (available e.g. from Sigma S-7388). Another example is Protazyme AK (azurine dyed crosslinked casein prepared as tablets by Megazyme T-PRAK). Example 2 of WO 01/58276 describes suitable protease assays. A preferred assay is the Protazyme assay of Example 2D (the pH and temperature should be adjusted to the protease in question as generally described previously).

The term “protease” is defined herein as an enzyme that hydrolyses peptide bonds. It includes any enzyme belonging to the EC 3.4 enzyme group (including each of the thirteen subclasses thereof http://en.wikipedia.Org/wiki/Category:EC_3.4). The EC number refers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, California, including supplements 1-5 published in Eur. J. Biochem. 1994, 223, 1-5; Eur. J. Biochem. 1995, 232, 1-6; Eur. J. Biochem. 1996, 237, 1-5; Eur. J. Biochem. 1997, 250, 1-6; and Eur. J. Biochem. 1999, 264, 610-650; respectively. The term "subtilases" refer to a sub-group of serine protease according to Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523. Serine proteases or serine peptidases is a subgroup of proteases characterized by having a serine in the active site, which forms a covalent adduct with the substrate. Further, the subtilases (and the serine proteases) are characterized by having two active site amino acid residues apart from the serine, namely a histidine and an aspartic acid residue. The subtilases may be divided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family.

A protease referred to herein may not only be natural or wildtype proteases, but also any mutants, variants, fragments etc. thereof exhibiting protease activity, as well as synthetic proteases, such as shuffled proteases, and consensus proteases. Such genetically engineered proteases can be prepared as is generally known in the art, e. g. by Site-directed Mutagenesis, by PCR (using a PCR fragment containing the desired mutation as one of the primers in the PCR reactions), or by Random Mutagenesis. The preparation of consensus proteins is described in e. g. EP 0 897 985.

Such non-wildtype proteases may be based on protease(s) derived from Nocardiopsis sp. NRRL 18262, and Nocardiopsis alba and have at least 60, 65, 70, 75, 80, 85, 90, or at least 95% amino acid identity but not 100% to a wildtype protease. For calculating percentage identity, any computer program known in the art can be used. Examples of such computer programs are the Clustal V algorithm (Higgins, D. G., and Sharp, P. M. (1989), Gene (Amsterdam), 73, 237-244 ; and the GAP program provided in the GCG version 8 program package (Program Manual for the Wisconsin Package, Version 8, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-453. In a particular embodiment, the protease referred to herein may advantageously be both, acid-stable and thermostable. The term "thermostable" means for proteases referred to herein to have a temperature optimum is at least 50 °C, 52 °C, 54 °C, 56 °C, 58 °C, 60 °C, 62 °C, 64 °C, 66 °C, °68 C, or at least °70 C.

The Figures show:

Figure 1 shows experimental feed compositions and in feeding trials (Ts).

Figure 2 shows a schematic view of protocol in gut microbiota composition analysis.

Figure 3 shows a schematic view of the methodologies used gut microbiota composition analysis.

Figure 4 shows growth performances of Litopenaeus vannamei fed diet containing different enzyme products (corresponding to T1-T6 of Figure 1) after 8 weeks feeding period.

Figure 5 shows feed utilization of Litopenaeus vannamei fed diet containing different enzyme products (corresponding to T1-T6 of Figure 1) after 8 weeks feeding period.

Figure 6 shows microbiome analysis in hepatopancreas of Pacific white shrimp Litopenaeus vannamei fed the experimental diets comprising a protease (SEQ ID NO. 1) for 8 weeks. 300 g protease per 1 ton of feed, have a reduced amount of Vibrio species while the amount of Lactobacillus species increased (see Figure 6, e.g., 1st bar (positive control with protease feed) as well as 2nd 3rd 5th and 6th bar (all with different amounts of protease feed) compared to 4th bar (negative control - without protease feed).

Note: “negative control” stands for a 15% fish meal diet; “positive control” stands for a 25% fishmeal diet, both negative and positive control are without protease; “ProAct” stands for the protease added to the fishmeal diet

Further note that “1” stands for Lactobacillus species, “2” stands for Gram-positive bacteria, “3” stands for Gram-negative bacteria, “4” stands for Vibrio species

Figure 7 shows the results from T1-T6 of Figure 1 as regards a decrease in deleterious bacteria, particularly Vibrio species. As of T3, shrimp were fed a composition comprising a protease (SEQ ID NO: 1). It is apparent that deleterious bacteria, particularly Vibrio species decrease when a protease is fed. The present invention may also be summarized in the following items:

(1) Use of a composition comprising a protease for improving the balance of beneficial and deleterious bacteria in the gastrointestinal tract of aquatic species.

(2) A method for improving the balance of beneficial and deleterious bacteria in the gastrointestinal tract of aqua species, comprising feeding aquatic species with a composition comprising a protease.

(3) A method of culturing an aquaculture of an aquatic species, comprising improving the balance of beneficial and deleterious bacteria in the gastrointestinal tract of said aquatic species by the means of a composition comprising a protease.

(4) The use of item 1 or the method of item 2 or 3, wherein improvement comprises an increase in the level of beneficial bacteria and a decrease in the level of deleterious bacteria.

(5) The use or the method of any one of the preceding items, wherein the beneficial bacteria comprise one or more of lactic acid bacteria, preferably Lactobacillus spp. and/or the deleterious bacteria comprise one or more of Vibrio ssp.

(6) The use or the method of any one of the preceding items, wherein the composition is a complete feed for aquatic species, a nutritional supplement, prebiotic or aquaculture inoculant.

(7) The use or the method of any one of the preceding items, wherein the composition further comprises a carrier.

(8) The use or the method of any one of the preceding items, wherein said composition further comprises one or more probiotic bacteria, preferably lactic acid bacteria, e.g. Lactobacillus spp.

(9) The use or the method of any one of the preceding items, wherein said aquatic species are selected from crustaceans or fish.

(10) The use or the method of item 9, wherein said fish are warm water fish or cold water fish.

(11) The use or the method of item 10, wherein said warm water fish are selected from tilapia, seabream, seabass, or carp.

(12) The use or the method of item 10, wherein said cold water fish are selected from salmon or rainbow trout. (13) The use or the method of item 9, wherein said crustaceans are shrimps.

(14) The use or the method of any one of the preceding items, wherein said protease is a serine protease.

(15) The use or the method of any one of the preceding items, wherein said protease is characterized in that it is

(a) a polypeptide having a sequence identity of at least 70% to any one of SEQ ID NOs 1-5;

(b) a variant of any one of SEQ ID NOs: 1-5, wherein the variant has protease activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions or any combination thereof in 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 positions;

(c) a polypeptide comprising the polypeptide of (a) or (b) and a N-terminal and/or C-terminal His-tag and/or HQ-tag;

(d) a polypeptide comprising the polypeptide of (a) or (b) and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; or

(e) a fragment of the polypeptide of (a) or (b) having protease activity and having at least 90% of the length of the mature polypeptide.

(16) The use or the method of any one of the preceding items, wherein said serine protease is Ronozyme ProAct.

(17) The use or the method of any one of the preceding items, wherein said serine protease is characterized in that it has the sequence shown in SEQ ID NO. 1 or 4.

(18) The use or method of any one of the preceding items, wherein said aquatic species are healthy.

(19) The use or method of any one of the preceding items, wherein said use or method is non-therapeutic Examples

The following examples illustrate the invention. These examples should not be construed as to limit the scope of this invention. The examples are included for purposes of illustration and the present invention is limited only by the claims.

Example 1 : Effect of different enzymes supplement on gut health in white shrimp

1. Test product

[001] Enzyme products from DSM, Ronozyme® Hiphos, Ronozyme® ProAct (SEQ ID NO: 1), Ronozyme® VP and HyD or ProAct 360 (SEQ ID NO: 4) can be used as feed additive for this trial. ProAct (SEQ ID NO: 1) was used in this trial.

2. Test animals

Pacific white shrimp (Litopenaeus vannamei) size 2-3 g were used for this trial. They were cultured from post larvae at AQST. Post larvae provided from a commercial shrimp farm in Thailand, and the pathogenic free status including WSSV, YHV, IHHNV, TSV, IMNV, AHPND and EHP will be checked by PCR in order to guarantee the shrimp health. Initially, shrimp were transferred to 390-L tank at salinity 15 ppt for 2 days prior to start the experiment.

3. Experimental design

The Completely Random Design experiment were set up as 6 treatments with 5 replicates containing 30 shrimp, size 2-3 g, in 390 L tanks as follows:

4. Experimental feeds and feeding

[002] The experimental feeds made by AQST and DSM containing 38% protein as shown in the Figure 1. The feeding rate at 5-10% bodyweight per day divided to 5 meals at 08h30, 11h30, 14h30, 17h00 and 21h00 of 8 weeks feeding period.

5. Laboratory analysis

5.1 Growth performance

This feeding trial conducted for 8 weeks. The individual shrimp weight and survival recorded at 0, 4 (sampling only 4-5 shrimp for weighing) and 8 week of feeding period. The feed intake recorded every day. The data collection processed for further calculation of feed conversion ratio, survival rate, average body weight, specific growth rate and average daily growth.

5.2 Gut microbiota composition

Gut microbiota examined at the end of feeding period (30 samples; pooled 5 shrimp/ sample I tank) for a total of 30 sample using next generation sequencing (Illumina Miseq) of 16S rRNA gene. Results were selected by 3 replicates and reported as relative abundance of bacterial groups, similarities of gut microbiota among different diets, and microbiota diversity indices.

5.3 Gut microbiota composition

Gut microbiota composition from shrimp tissue samples from DSM was analyzed according to the following scheme (Figure 2). Intestine microbiome analysis was carried out according to the methodology depicted in Figure 3.

6. Results

Growth performances of Litopenaeus vannamei fed diet containing different enzyme products after 8 weeks feeding period (Figure 4).

Feed utilization of Litopenaeus vannamei fed diet containing different enzyme products after 8 weeks feeding period (Figure 5).

Overall, in this trial, a reduction of deleterious bacteria, particularly Vibrio species was observed when shrimp were fed with a composition comprising protease (SEQ ID NO: 1) in an amount of 300g per ton feed. As can be seen from Figure 7, in the treatments T3 to T6, there is a decrease in the level of deleterious bacteria, particularly Vibrio species. This is illustrated by the arrow starting at the left edge and going down to the right edge. Nearly all species below the arrow are Vibrio species.

Example 2: Microbiome analysis in hepatopancreas of Pacific white shrimp (Litopenaeus vannamei) fed the experimental diets for 8 weeks

In this trial microbiome analysis was carried out in hepatopancreas of Pacific white shrimp (Litopenaeus vannamei) an experimental diet for 8 weeks (Figure 6). Accordingly, Figure 6 shows that shrimps fed with a protease (SEQ ID NO: 1), e.g., 300 g protease per 1 ton of feed, have a reduced amount of Vibrio species while the amount of Lactobacillus species increased; see Figure 6, e.g., 1^st bar (positive control) as well as 2^nd 3^rd 5^th and 6^th bar (all with different amounts of protease feed) compared to 4^th bar.

The trend is that the addition of a protease increases the level of beneficial bacteria, particularly Lactobacillus species, while at the same time the level of deleterious bacteria, e.g. Vibrio species decreases. Indeed, as soon as protease is added, the effect occurs, i.e., the balance between the level of beneficial bacteria and deleterious bacteria is improved.

Note: “negative control” stands for a 15% fish meal diet; “positive control” stands for a 25% fishmeal diet, both negative and positive control are without protease; “ProAct” stands for the protease added to the fishmeal diet. ***

One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Further, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The compositions, methods, procedures, treatments, molecules and specific compounds described herein are presently representative of certain embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims. The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions embodied herein may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. All documents, including patent applications and scientific publications, referred to herein are incorporated herein by reference for all purposes.

Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

Claims Use of a composition comprising a protease for improving the balance of beneficial and deleterious bacteria in the gastrointestinal tract of aquatic species. A method for improving the balance of beneficial and deleterious bacteria in the gastrointestinal tract of aqua species, comprising feeding aquatic species with a composition comprising a protease. A method of culturing an aquaculture of an aquatic species, comprising improving the balance of beneficial and deleterious bacteria in the gastrointestinal tract of said aquatic species by the means of a composition comprising a protease. The use of claim 1 or the method of claim 2 or 3, wherein improvement comprises an increase in the level of beneficial bacteria and a decrease in the level of deleterious bacteria. The use or the method of any one of the preceding claims, wherein the beneficial bacteria comprise one or more of lactic acid bacteria, preferably Lactobacillus spp. and/or the deleterious bacteria comprise one or more of Vibrio ssp. The use or the method of any one of the preceding claims, wherein the composition is a complete feed for aquatic species, a nutritional supplement, prebiotic or aquaculture inoculant. The use or the method of any one of the preceding claims, wherein the composition further comprises a carrier. The use or the method of any one of the preceding claims, wherein said aquatic species are selected from crustaceans or fish. The use or the method of claim 8, wherein said fish are warm water fish or cold water fish. The use or the method of claim 8, wherein said crustaceans are shrimps. The use or the method of any one of the preceding claims, wherein said protease is a serine protease. The use or the method of any one of the preceding claims, wherein said protease is characterized in that it is (a) a polypeptide having a sequence identity of at least 70% to any one of SEQ ID NOs 1-5;

(e) a fragment of the polypeptide of (a) or (b) having protease activity and having at least 90% of the length of the mature polypeptide. The use or the method of any one of the preceding claims, wherein said serine protease is Ronozyme ProAct. The use or the method of any one of the preceding claims, wherein said serine protease is characterized in that it has the sequence shown in SEQ ID NO. 1 or 4. The use or method of any one of the preceding claims, wherein said aquatic species are healthy.